Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition.
Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition.
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DOI：
10.1371/journal.pone.0017916
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发表时间：
2011-03-23
期刊：
影响因子：
3.7
通讯作者：
Wellington EM
中科院分区：
文献类型：
--
作者：
Pontiroli A;Travis ER;Sweeney FP;Porter D;Gaze WH;Mason S;Hibberd V;Holden J;Courtenay O;Wellington EM
牛结核病是近年来在英国发病率显著上升的一种重要的复发性人畜共患病，其病原体是牛分枝杆菌。已经确定了这种疾病的野生动物宿主，但对牛的传播方式仍不清楚。有证据表明，可行的M。牛的细胞可以在土壤和粪便中存活一年以上。我们报告了一项多操作员盲法试验，对从各种土壤和粪便样品中提取DNA的五种方法进行严格比较，以评估M的回收率。牛血清中的DNA进行实时荧光PCR检测。这些方法包括四种商业试剂盒：具有预处理步骤的QIAamp Stool Mini试剂盒、FastDNA® Spin试剂盒、UltraClean™和PowerSoil™污垢试剂盒以及基于苯酚：氯仿纯化的已发表的手动方法，称为Griffiths。M.由四名操作员提取牛BCG巴斯德加标样品，并使用特异性实时PCR测定进行评价。一种新的抑制对照测定法与分光光度比一起用于监测影响PCR、DNA产率和纯度的抑制化合物的水平。M.提取方法和环境样本类型之间的牛检测;操作员之间未观察到显著差异。还评估了处理时间和成本。为了提高M.牛检测进一步优化了两种性能最佳的方法，FastDNA® Spin试剂盒和Griffiths，并采用了ABI TaqMan环境PCR Master mix，从而提高了灵敏度。 M.在所有环境样品中均成功检测到牛;使用FastDNA® Spin试剂盒提取DNA是最灵敏的方法，在所有测试的土壤类型中回收率最高。对于麻烦的粪便样品，我们使用并推荐了一种基于体积减小的改进检测方法，Griffiths的检测限为4.25×105个细胞g−1，FastDNA® Spin试剂盒的检测限为4.25×106个细胞g−1。
Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year. We report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities. M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×105 cells g−1 using Griffiths and 4.25×106 cells g−1 using FastDNA® Spin kit.
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影响因子：
1.9
作者：
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通讯作者：
COPENHAVER, MD
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